RESUMO
BACKGROUND: Hypoglycemia occurs frequently in the neonate and may result in neurologic dysfunction. Its impact on the kinetics of cellular respiration and bioenergetics in the neonatal brain remains to be explored. AIMS: Develop murine model to investigate the effects of hypoglycemia on neonatal brain bioenergetics. STUDY DESIGN: Forebrain fragments were excised from euthanized BALB/c pups aged <24 hours to 14 days. We measured cellular respiration (µM O2 min-1.mg-1) in phosphate-buffered saline with and without glucose, using phosphorescence oxygen analyzer, as well as cellular adenosine triphosphate (ATP, nmol.mg-1) using the luciferin-luciferase system. RESULTS: In the presence of glucose, although cellular respiration was 11% lower in pups ≤3 days compared to those 3- 14 days old (0.48 vs. 0.54), that difference was not statistically significant (pâ=â0.14). Respiration driven by endogenous metabolic fuels (without added glucose) was 16% lower in pups ≤3 days compared to those 3- 14 days (0.35 vs. 0.42, pâ=â0.03), confirming their increased dependency on exogenous glucose. Although cellular ATP was similar between the two age groups (14.9 vs. 11.2, pâ=â0.32), the ATP content was more severely depleted without added glucose in the younger pups, especially in the presence of the cytochrome c oxidase inhibitor cyanide. The first-order rate constant of cellular ATP decay (hydrolysis) was 44% lower in 2-day-old pups compared to 14-day-old mice (0.43 vs. 0.77 min-1, pâ=â0.03). CONCLUSIONS: Forebrain cellular respiration and ATP consumption are lower in young pups than older mice. In the absence of glucose, the support for these processes is reduced in young pups, explaining their brain hypersensitivity to hypoglycemia.
Assuntos
Trifosfato de Adenosina/metabolismo , Animais Recém-Nascidos/fisiologia , Metabolismo Energético , Hipoglicemia/fisiopatologia , Consumo de Oxigênio/efeitos dos fármacos , Prosencéfalo/fisiopatologia , Fatores Etários , Animais , Respiração Celular/efeitos dos fármacos , Modelos Animais de Doenças , Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Glucose/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Prosencéfalo/metabolismo , Cianeto de Sódio/farmacologiaRESUMO
We previously reported that KW-2449, (E)-1-{4-[2-(1H-Indazol-3-yl)vinyl]benzoyl}piperazine, a novel multikinase inhibitor developed for the treatment of leukemia patients, was oxidized to an iminium ion intermediate by monoamine oxidase B (MAO-B) and then converted to its oxo-piperazine form (M1) by aldehyde oxidase (AO). However, it was found that the significant decrease in the pharmacologically active metabolite M1 following repeated administration of KW-2449 in primates might hamper the effectiveness of the drug. The mechanism underlying this phenomenon was investigated and it was found that the AO activity was inhibited in a time-dependent manner in vitro under the co-incubation of KW-2449 and MAO-B, while neither KW-2449 nor M1 strongly inhibited MAO-B or AO activity. These results clearly suggest that MAO-B catalysed iminium ion metabolite inhibited AO, prompting us to investigate whether or not the iminium ion metabolite covalently binds to endogenous proteins, as has been reported with other reactive metabolites as a cause for idiosyncratic toxicity. The association of the radioactivity derived from 14 C-KW-2449 with endogenous proteins both in vivo and in vitro was confirmed and it was verified that this covalent binding was inhibited by the addition of sodium cyanide, an iminium ion-trapping reagent, and pargyline, a MAO-B inhibitor. These findings strongly suggest that the iminium ion metabolite of KW-2449 is highly reactive in inhibiting AO irreversibly and binding to endogenous macromolecules covalently.
Assuntos
Aldeído Oxidase/antagonistas & inibidores , Indazóis/metabolismo , Indazóis/farmacologia , Piperazinas/metabolismo , Piperazinas/farmacologia , Proteínas/metabolismo , Aldeído Oxidase/metabolismo , Animais , Isótopos de Carbono , Humanos , Macaca fascicularis , Monoaminoxidase/metabolismo , Inibidores da Monoaminoxidase/farmacologia , Oxirredução , Pargilina/farmacologia , Ligação Proteica , Ensaio Radioligante , Cianeto de Sódio/farmacologiaRESUMO
The application of sodium cyanide (NaCN) to the carotid body receptors (CBR) (CBR stimulation) induces rapid blood hyperglycemia and an increase in brain glucose retention. The commissural nucleus tractus solitarius (cNTS) is an essential relay nucleus in this hyperglycemic reflex; it receives glutamatergic afferents (that also release brain derived neurotrophic factor, BDNF) from the nodose-petrosal ganglia that relays CBR information. Previous work showed that AMPA in NTS blocks hyperglycemia and brain glucose retention after CBR stimulation. In contrast, BDNF, which attenuates glutamatergic AMPA currents in NTS, enhances these glycemic responses. Here we investigated the combined effects of BDNF and AMPA (and their antagonists) in NTS on the glycemic responses to CBR stimulation. Microinjections of BDNF plus AMPA into the cNTS before CBR stimulation in anesthetized rats, induced blood hyperglycemia and an increase in brain arteriovenous (a-v) of blood glucose concentration difference, which we infer is due to increased brain glucose retention. By contrast, the microinjection of the TrkB antagonist K252a plus AMPA abolished the glycemic responses to CBR stimulation similar to what is observed after AMPA pretreatments. In BDNF plus AMPA microinjections preceding CBR stimulation, the number of c-fos immunoreactive cNTS neurons increased. In contrast, in the rats microinjected with K252a plus AMPA in NTS, before CBR stimulation, c-fos expression in cNTS decreased. The expression of AMPA receptors GluR2/3 did not change in any of the studied groups. These results indicate that BDNF in cNTS plays a key role in the modulation of the hyperglycemic reflex initiated by CBR stimulation.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Corpo Carotídeo/efeitos dos fármacos , Corpo Carotídeo/metabolismo , Hiperglicemia/metabolismo , Núcleo Solitário/metabolismo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/administração & dosagem , Ensaio de Imunoadsorção Enzimática , Glucose/metabolismo , Hiperglicemia/induzido quimicamente , Hiperglicemia/patologia , Imuno-Histoquímica , Masculino , Microinjeções , Neurotransmissores/farmacologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Distribuição Aleatória , Ratos Wistar , Receptor trkB/agonistas , Receptor trkB/antagonistas & inibidores , Receptor trkB/metabolismo , Receptores de AMPA/agonistas , Receptores de AMPA/antagonistas & inibidores , Receptores de AMPA/metabolismo , Cianeto de Sódio/farmacologia , Núcleo Solitário/citologia , Núcleo Solitário/efeitos dos fármacos , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/administração & dosagemRESUMO
The Parkinson's disease (PD)-related protein F-box only protein 7 (Fbxo7) is the substrate-recognition component of the Skp1-Cullin-F-box protein E3 ubiquitin ligase complex. We have recently shown that PD-associated mutations in Fbxo7 disrupt mitochondrial autophagy (mitophagy), suggesting a role for Fbxo7 in modulating mitochondrial homeostasis. Here we report that Fbxo7 deficiency is associated with reduced cellular NAD+ levels, which results in increased mitochondrial NADH redox index and impaired activity of complex I in the electron transport chain. Under these conditions of compromised respiration, mitochondrial membrane potential and ATP contents are reduced, and cytosolic reactive oxygen species (ROS) production is increased. ROS activates poly (ADP-ribose) polymerase (PARP) activity in Fbxo7-deficient cells. PARP inhibitor restores cellular NAD+ content and redox index and ATP pool, suggesting that PARP overactivation is cause of decreased complex I-driven respiration. These findings bring new insight into the mechanism of Fbxo7 deficiency, emphasising the importance of mitochondrial dysfunction in PD.
Assuntos
Proteínas F-Box/metabolismo , Mitocôndrias/metabolismo , Doença de Parkinson/patologia , Poli(ADP-Ribose) Polimerases/metabolismo , Trifosfato de Adenosina/metabolismo , Células Cultivadas , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Complexo I de Transporte de Elétrons/metabolismo , Proteínas F-Box/antagonistas & inibidores , Proteínas F-Box/genética , Humanos , Ácido Iodoacético/farmacologia , Isoquinolinas/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitofagia/efeitos dos fármacos , NAD/química , NAD/metabolismo , Consumo de Oxigênio , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Piperidinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/química , Polimorfismo de Nucleotídeo Único , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Cianeto de Sódio/farmacologiaRESUMO
A recent study showed that hypoxia activates a Ca2+-sensitive, Na+-permeable non-selective cation channel (NSC) in carotid body glomus cells. We studied the effects of mitochondrial inhibitors that increase Ca2+ influx via Ca2+ channel (Cav), and receptor agonists that release Ca2+ from endoplasmic reticulum (ER) on NSC. Mitochondrial inhibitors (NaCN, FCCP, H2S, NO) elevated [Ca2+]i and activated NSC. Angiotensin II and acetylcholine that elevate [Ca2+]i via the Gq-IP3 pathway activated NSC. However, endothelin-1 (Gq) and 5-HT (Gq) showed little or no effect on [Ca2+]i and did not activate NSC. Adenosine (Gs) caused a weak rise in [Ca2+]i but did not activate NSC. Dopamine (Gs) and γ-aminobytyric acid (Gi) were ineffective in raising [Ca2+]i and failed to activate NSC. Store-operated Ca2+ entry (SOCE) produced by depletion of Ca2+ stores with cyclopiazonic acid activated NSC. Our results show that Ca2+ entry via Cav, ER Ca2+ release and SOCE can activate NSC. Thus, NSC contributes to both voltage- and receptor-mediated excitation of glomus cells.
Assuntos
Cálcio/metabolismo , Corpo Carotídeo/citologia , Células Quimiorreceptoras/fisiologia , Canais Iônicos/metabolismo , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Angiotensina II/farmacologia , Anilidas/farmacologia , Animais , Animais Recém-Nascidos , Cafeína/farmacologia , Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Células Quimiorreceptoras/efeitos dos fármacos , Relação Dose-Resposta a Droga , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Inibidores Enzimáticos/farmacologia , Hipóxia/fisiopatologia , Ionóforos de Próton/farmacologia , Ratos , Ratos Sprague-Dawley , S-Nitroso-N-Acetilpenicilamina/farmacologia , Cianeto de Sódio/farmacologia , Tiadiazóis/farmacologiaRESUMO
Eyes are supplied O2 through the cornea and vessels of the retina and iris, which are tissues characterized by aerobic metabolism. Meanwhile, there are no reports on the association between iris sphincter contraction and aerobic metabolism. In this paper, we studied the aforementioned association. Eyes from adult pigs of either sex were obtained from a local abattoir. A muscle strip was connected to a transducer to isometrically record the tension. O2 consumption was measured using a Clark-type polarograph connected to a biological oxygen monitor. Creatine phosphate (PCr) and adenosine triphosphate (ATP) contents were measured in the muscle strips by high-performance liquid chromatography (HPLC). Iris sphincter muscles were measured in resting, contractile or hypoxic phases. Contraction was induced by hyperosmotic 65 mM KCl (H-65K+) or carbachol (CCh), and hypoxia was induced by aeration with N2 instead of O2 or by addition of sodium cyanide (NaCN). H-65K+- and CCh-induced muscle contraction, involved increasing O2 consumption. Hypoxia and NaCN significantly decreased H-65K+- and CCh-induced muscle contraction and/or O2 consumption and PCr contents. Our results suggest that the contractile behavior in porcine iris sphincter highly depends on mitogen oxidative metabolism.
Assuntos
Iris/metabolismo , Músculo Liso/metabolismo , Consumo de Oxigênio/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Carbacol/farmacologia , Hipóxia Celular , Feminino , Iris/efeitos dos fármacos , Masculino , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Músculo Liso/efeitos dos fármacos , Fosfocreatina/metabolismo , Cloreto de Potássio/farmacologia , Cianeto de Sódio/farmacologia , SuínosRESUMO
Global or local ischemia contributes to the pathogenesis of acute kidney injury (AKI). Currently there are no specific therapies to prevent AKI. Potentiation of glycolytic metabolism and attenuation of mitochondrial respiration may decrease cell injury and reduce reactive oxygen species generation from the mitochondria. Meclizine, an over-the-counter anti-nausea and -dizziness drug, was identified in a 'nutrient-sensitized' chemical screen. Pretreatment with 100 mg/kg of meclizine, 17 h prior to ischemia protected mice from IRI. Serum creatinine levels at 24 h after IRI were 0.13 ± 0.06 mg/dl (sham, n = 3), 1.59 ± 0.10 mg/dl (vehicle, n = 8) and 0.89 ± 0.11 mg/dl (meclizine, n = 8). Kidney injury was significantly decreased in meclizine treated mice compared with vehicle group (p < 0.001). Protection was also seen when meclizine was administered 24 h prior to ischemia. Meclizine reduced inflammation, mitochondrial oxygen consumption, oxidative stress, mitochondrial fragmentation, and tubular injury. Meclizine preconditioned kidney tubular epithelial cells, exposed to blockade of glycolytic and oxidative metabolism with 2-deoxyglucose and NaCN, had reduced LDH and cytochrome c release. Meclizine upregulated glycolysis in glucose-containing media and reduced cellular ATP levels in galactose-containing media. Meclizine inhibited the Kennedy pathway and caused rapid accumulation of phosphoethanolamine. Phosphoethanolamine recapitulated meclizine-induced protection both in vitro and in vivo.
Assuntos
Precondicionamento Isquêmico , Rim/irrigação sanguínea , Rim/patologia , Meclizina/uso terapêutico , Substâncias Protetoras/uso terapêutico , Traumatismo por Reperfusão/tratamento farmacológico , Injúria Renal Aguda/complicações , Injúria Renal Aguda/tratamento farmacológico , Injúria Renal Aguda/patologia , Trifosfato de Adenosina/metabolismo , Animais , Respiração Celular/efeitos dos fármacos , Citocromos c/metabolismo , Desoxiglucose/farmacologia , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Etanolaminas/metabolismo , Galactose/farmacologia , Glicólise/efeitos dos fármacos , Humanos , Inflamação/complicações , Inflamação/patologia , Rim/efeitos dos fármacos , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/metabolismo , Túbulos Renais/patologia , L-Lactato Desidrogenase/metabolismo , Células LLC-PK1 , Masculino , Meclizina/farmacologia , Meclizina/toxicidade , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Substâncias Protetoras/farmacologia , Traumatismo por Reperfusão/complicações , Traumatismo por Reperfusão/patologia , Cianeto de Sódio/farmacologia , Suínos , Regulação para Cima/efeitos dos fármacosRESUMO
Disruption of the blood-brain barrier (BBB) integrity occurring during the early onset of stroke is not only a consequence of, but also contributes to the further progression of stroke. Although it has been well documented that brain microvascular endothelial cells and astrocytes play a critical role in the maintenance of BBB integrity, pericytes, sandwiched between endothelial cells and astrocytes, remain poorly studied in the pathogenesis of stroke. Our findings demonstrated that treatment of human brain microvascular pericytes with sodium cyanide (NaCN) and glucose deprivation resulted in increased expression of vascular endothelial growth factor (VEGF) via the activation of tyrosine kinase Src, with downstream activation of mitogen activated protein kinase and PI3K/Akt pathways and subsequent translocation of NF-κB into the nucleus. Conditioned medium from NaCN-treated pericytes led to increased permeability of endothelial cells, and this effect was significantly inhibited by VEGF-neutralizing antibody. The in vivo relevance of these findings was further corroborated in the stroke model of mice wherein the mice, demonstrated disruption of the BBB integrity and concomitant increase in the expression of VEGF in the brain tissue as well as in the isolated microvessel. These findings thus suggest the role of pericyte-derived VEGF in modulating increased permeability of BBB during stroke. Understanding the regulation of VEGF expression could open new avenues for the development of potential therapeutic targets for stroke and other neurological disease.
Assuntos
Barreira Hematoencefálica , Endotélio Vascular/citologia , Pericitos/citologia , Acidente Vascular Cerebral/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Células Cultivadas , Endotélio Vascular/metabolismo , Genes src , Humanos , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pericitos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Cianeto de Sódio/farmacologia , Acidente Vascular Cerebral/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
The effective integrated organization of processes in cardiac cells is achieved, in part, by the functional compartmentation of energy transfer processes. Earlier, using permeabilized cardiomyocytes, we demonstrated the existence of tight coupling between some of cardiomyocyte ATPases and glycolysis in rat. In this work, we studied contribution of two membrane ATPases and whether they are coupled to glycolysis--sarcoplasmic reticulum Ca2+ ATPase (SERCA) and plasmalemma Na+/K+-ATPase (NKA). While SERCA activity was minor in this preparation in the absence of calcium, major role of NKA was revealed accounting to â¼30% of the total ATPase activity which demonstrates that permeabilized cell preparation can be used to study this pump. To elucidate the contribution of NKA in the pool of ATPases, a series of kinetic measurements was performed in cells where NKA had been inhibited by 2 mM ouabain. In these cells, we recorded: ADP- and ATP-kinetics of respiration, competition for ADP between mitochondria and pyruvate kinase (PK), ADP-kinetics of endogenous PK, and ATP-kinetics of total ATPases. The experimental data was analyzed using a series of mathematical models with varying compartmentation levels. The results show that NKA is tightly coupled to glycolysis with undetectable flux of ATP between mitochondria and NKA. Such tight coupling of NKA to PK is in line with its increased importance in the pathological states of the heart when the substrate preference shifts to glucose.
Assuntos
Glicólise , Miócitos Cardíacos/metabolismo , ATPase Trocadora de Sódio-Potássio/fisiologia , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Transporte Biológico Ativo , Compartimento Celular , Membrana Celular/enzimologia , Permeabilidade da Membrana Celular , Feminino , L-Lactato Desidrogenase/metabolismo , Masculino , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Modelos Cardiovasculares , Oligomicinas/farmacologia , Ouabaína/farmacologia , Consumo de Oxigênio/efeitos dos fármacos , Piruvato Quinase/metabolismo , Ratos , Ratos Wistar , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/antagonistas & inibidores , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/fisiologia , Cianeto de Sódio/farmacologia , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Tapsigargina/farmacologiaRESUMO
The metabolic hypothesis of carotid body chemoreceptor hypoxia transduction proposes an impairment of ATP production as the signal for activation. We hypothesized that mitochondrial complex IV blockers and hypoxia would act synergistically in exciting afferent nerve activity. Following a pre-treatment with low dosage sodium cyanide (10-20µM), the hypoxia-induced nerve response was significantly reduced along with hypoxia-induced catecholamine release. However, in isolated glomus cells, the intracellular calcium response was enhanced as initially predicted. This suggests a cyanide-mediated impairment in the step between the glomus cell intracellular calcium rise and neurotransmitter release from secretory vesicles. Administration of a PKC blocker largely reversed the inhibitory actions of cyanide on the neural response. We conclude that the expected synergism between cyanide and hypoxia occurs at the level of glomus cell intracellular calcium but not at downstream steps due to a PKC-dependent inhibition of secretion. This suggests that at least one regulatory step beyond the glomus cell calcium response may modulate the magnitude of chemoreceptor responsiveness.
Assuntos
Corpo Carotídeo/citologia , Corpo Carotídeo/metabolismo , Corpo Carotídeo/fisiopatologia , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Inibidores Enzimáticos/farmacologia , Hipóxia/metabolismo , Potenciais de Ação/efeitos dos fármacos , Trifosfato de Adenosina/farmacologia , Análise de Variância , Animais , Animais Recém-Nascidos , Cálcio/metabolismo , Catecolaminas/metabolismo , Células Quimiorreceptoras/efeitos dos fármacos , Células Quimiorreceptoras/fisiologia , Relação Dose-Resposta a Droga , Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Técnicas de Patch-Clamp , Ratos , Cianeto de Sódio/farmacologiaRESUMO
Borrelia burgdorferi, the causative agent of Lyme disease, exists in nature through a complex life cycle involving ticks of the Ixodes genus and mammalian hosts. During its life cycle, B. burgdorferi experiences fluctuations in oxygen tension and may encounter reactive oxygen species (ROS). The key metalloenzyme to degrade ROS in B. burgdorferi is SodA. Although previous work suggests that B. burgdorferi SodA is an iron-dependent superoxide dismutase (SOD), later work demonstrates that B. burgdorferi is unable to transport iron and contains an extremely low intracellular concentration of iron. Consequently, the metal cofactor for SodA has been postulated to be manganese. However, experimental evidence to support this hypothesis remains lacking. In this study, we provide biochemical and genetic data showing that SodA is a manganese-dependent enzyme. First, B. burgdorferi contained SOD activity that is resistant to H(2)O(2) and NaCN, characteristics associated with Mn-SODs. Second, the addition of manganese to the Chelex-treated BSK-II enhanced SodA expression. Third, disruption of the manganese transporter gene bmtA, which significantly lowers the intracellular manganese, greatly reduced SOD activity and SodA expression, suggesting that manganese regulates the level of SodA. In addition, we show that B. burgdorferi is resistant to streptonigrin, a metal-dependent redox cycling compound that produces ROS, and that SodA plays a protective role against the streptonigrin. Taken together, our data demonstrate the Lyme disease spirochete encodes a manganese-dependent SOD that contributes to B. burgdorferi defense against intracellular superoxide.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Proteínas de Bactérias/metabolismo , Borrelia burgdorferi/enzimologia , Farmacorresistência Bacteriana/fisiologia , Ferro , Estreptonigrina/farmacologia , Superóxido Dismutase/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Borrelia burgdorferi/genética , Farmacorresistência Bacteriana/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Transporte de Íons/efeitos dos fármacos , Transporte de Íons/fisiologia , Oxidantes/farmacologia , Cianeto de Sódio/farmacologia , Hidróxido de Sódio/farmacologia , Superóxido Dismutase/antagonistas & inibidores , Superóxido Dismutase/genética , Superóxidos/metabolismoRESUMO
It has been known that the inhibition of mitochondrial cytochrome c oxidase is one of the earliest events occurring under hypoxia and this inhibition can lead to neuronal damages. Thus, the cytochrome c oxidase inhibitor sodium cyanide (NaCN) is widely used to produce a model of chemical hypoxia by inhibiting this enzyme. However, the downstream signaling pathways of the inhibition of the cytochrome c oxidase remain to be studied. In the present paper, we used sodium cyanide to mimic the inhibition of the mitochondrial cytochrome c oxidase and studied its effect on glutamate release in synaptosomes from the prefrontal cortex using on-line fluorimetry. We also further investigated the mechanisms underlying the enhancing effect of sodium cyanide on glutamate release using pharmacological approaches combined with other techniques. The results showed that sodium cyanide significantly increased glutamate release from synaptosomes of prefrontal cortex; the broad-spectrum free radical scavenger MnTBAP and melatonin completely abolished the effect of sodium cyanide on glutamate release; the H2O2-NMDA receptor pathway mediated one part, whereas the lipid peroxyl radicals-ATP synthase pathway mediated another part of the sodium cyanide-induced glutamate release; scavenging H2O2 and enhancing ATP synthase activity could completely abolish the sodium cyanide-induced glutamate release.
Assuntos
Radicais Livres/metabolismo , Ácido Glutâmico/metabolismo , Hipóxia/metabolismo , Córtex Pré-Frontal/metabolismo , Sinaptossomos/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Canais de Cálcio/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Inibidores Enzimáticos/farmacologia , Peróxido de Hidrogênio/metabolismo , Hipóxia/induzido quimicamente , Peroxidação de Lipídeos , Masculino , ATPases Mitocondriais Próton-Translocadoras/antagonistas & inibidores , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Oxidantes/metabolismo , Córtex Pré-Frontal/ultraestrutura , Proteína Quinase C/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/metabolismo , Transdução de Sinais/fisiologia , Cianeto de Sódio/farmacologia , Sinaptossomos/efeitos dos fármacos , Valina/análogos & derivados , Valina/metabolismoRESUMO
BACKGROUND: There has been an ongoing debate in the reproductive field about whether mammalian spermatozoa rely on glycolysis, oxidative phosphorylation or both for their energy production. Recent studies have proposed that human spermatozoa depend mainly on glucose for motility and fertilization but the mechanism behind an efficient glycolysis in human spermatozoa is not well understood. Here, we demonstrate how human spermatozoa utilize exogenous pyruvate to enhance glycolytic ATP production, motility, hyperactivation and capacitation, events that are crucial for male fertility. METHODS: Purified human spermatozoa from healthy donors were incubated under capacitating conditions (including albumin, bicarbonate and glucose) and tested for changes in ATP levels, motility, hyperactivation and tyrosine phosphorylation after treatment with pyruvate. The experiments were repeated in the presence of sodium cyanide in order to assess the contribution from mitochondrial respiration. The metabolism of (13)C labeled glucose and pyruvate was traced by a combination of liquid chromatography and mass spectrometry. RESULTS: The treatment of human spermatozoa with exogenous pyruvate increased intracellular ATP levels, progressive motility and hyperactivation by 56, 21 and 130%, respectively. In addition, added pyruvate induced a significant increase in tyrosine phosphorylation levels. Blocking of the electron transport chain did not markedly affect the results, indicating that the mechanism is independent of oxidative phosphorylation. However, the observed effects could be counteracted by oxamate, an inhibitor of lactate dehydrogenase (LDH). Metabolic tracing experiments revealed that the observed rise in ATP concentration resulted from an enhanced glycolytic flux, which was increased by more than 50% in the presence of exogenous pyruvate. Moreover, all consumed (13)C labeled pyruvate added was converted to lactate rather than oxidized in the tricarboxylic acid cycle. CONCLUSIONS: Human spermatozoa seem to rely mainly, if not entirely, on glycolysis as the source of ATP fueling the energy-demanding processes of motility and capacitation. The efficient glycolysis is dependent on exogenous pyruvate, which indirectly feeds the accelerated glycolysis with NAD(+) through the LDH-mediated conversion of pyruvate to lactate. Pyruvate is present in the human female reproductive tract at concentrations in accordance with our results. As seen in other mammals, the motility and fertility of human spermatozoa seem to be dictated by the available energy substrates present in the conspecific female.
Assuntos
Ácido Pirúvico/farmacologia , Capacitação Espermática/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Glicólise/efeitos dos fármacos , Humanos , Ácido Láctico/metabolismo , Ácido Láctico/farmacologia , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fosforilação , Cianeto de Sódio/farmacologia , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Espermatozoides/fisiologia , Tirosina/metabolismoRESUMO
This study examined the inhibitory effects of papaverine on twitches directly elicited by electrical stimulation of the mouse diaphragm. Papaverine (3-100 µM) inhibited twitches in a dose-dependent manner. Papaverine increased the cyclic adenosine monophosphate (cAMP) but not cyclic guanosine monophosphate (cGMP) content. IBMX, Db-cAMP and 8-br-cGMP did not affect twitches, whereas verapamil and NaCN inhibited twitches. Increases in extracellular Ca²+ removed the twitch inhibition caused by verapamil but not that caused by papaverine. Papaverine (30 and 100 µM) and NaCN (1 mM) decreased creatine phosphate and ATP contents. These results suggest that the relaxing effects of papaverine on mouse diaphragm are mainly due to inhibition of aerobic energy metabolism.
Assuntos
Diafragma/efeitos dos fármacos , Contração Muscular/efeitos dos fármacos , Papaverina/farmacologia , Vasodilatadores/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Diafragma/metabolismo , Relação Dose-Resposta a Droga , Estimulação Elétrica , Masculino , Camundongos , Papaverina/administração & dosagem , Fosfocreatina/efeitos dos fármacos , Fosfocreatina/metabolismo , Cianeto de Sódio/farmacologia , Vasodilatadores/administração & dosagemRESUMO
We propose a simple method for producing micropatterned cell spots by photocatalytic lithography on a Pt porphyrin-based oxygen-sensitive polystyrene membrane that enables real-time imaging of oxygen consumption of patterned cell spots with sub-millimetre resolution.
Assuntos
Membranas Artificiais , Oxigênio/metabolismo , Análise Serial de Tecidos/métodos , Respiração Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células Hep G2 , Humanos , Microscopia de Fluorescência , Poliestirenos/química , Porfirinas/química , Cianeto de Sódio/farmacologiaRESUMO
We evaluate in rats the role of NO in the solitary tract nucleus (STn) after an anoxic stimulus to carotid body chemoreceptor cells (CChrc) with cyanide (NaCN), on the hyperglycemic reflex with glucose retention by the brain (BGR) and FOS expression (FOS-ir) in the STn. The results suggest that nitroxidergic pathways in the STn may play an important role in glucose homeostasis. A NO donor such as sodium nitroprusside (NPS) in the STn before CChrc stimulation increased arterial glucose level and significantly decreased BGR. NPS also induced a higher FOS-ir expression in STn neurons when compared to neurons in control rats that only received artificial cerebrospinal fluid (aCSF) before CChrc stimulation. In contrast, a selective NOS inhibitor such as Nomega-nitro-L-arginine methyl ester (L-NAME) in the STn before CChrc stimulation resulted in an increase of both, systemic glucose and BGR above control values. In this case, the number of FOS-ir positive neurons in the STn decreased when compared to control or to NPS experiments. FOS-ir expression in brainstem cells suggests that CChrc stimulation activates nitroxidergic pathways in the STn to regulate peripheral and central glucose homeostasis. The study of these functionally defined cells will be important to understand brain glucose homeostasis.
Assuntos
Corpo Carotídeo/metabolismo , Regulação da Expressão Gênica , Glucose/metabolismo , Homeostase , Óxido Nítrico/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Núcleo Solitário/metabolismo , Animais , Corpo Carotídeo/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Masculino , Neurônios/metabolismo , Nitroprussiato/farmacologia , Fotomicrografia , Ratos , Ratos Wistar , Cianeto de Sódio/farmacologia , Núcleo Solitário/efeitos dos fármacos , Fatores de TempoRESUMO
AIM: Hydrogen peroxide (H2O2) is produced during liver transplantation. Ischemia/reperfusion induces oxidation and causes intracellular Ca2+ overload, which harms liver cells. Our goal was to determine the precise mechanisms of these processes. METHODS: Hepatocytes were extracted from rats. Intracellular Ca2+ concentrations ([Ca2+](i)), inner mitochondrial membrane potentials and NAD(P)H levels were measured using fluorescence imaging. Phospholipase C (PLC) activity was detected using exogenous PIP2. ATP concentrations were measured using the luciferin-luciferase method. Patch-clamp recordings were performed to evaluate membrane currents. RESULTS: H2O2 increased intracellular Ca2+ concentrations ([Ca2+](i)) across two kinetic phases. A low concentration (400 micromol/L) of H2O2 induced a sustained elevation of [Ca2+](i) that was reversed by removing extracellular Ca2+. H2O2 increased membrane currents consistent with intracellular ATP concentrations. The non-selective ATP-sensitive cation channel blocker amiloride inhibited H2O2-induced membrane current increases and [Ca2+](i) elevation. A high concentration (1 mmol/L)of H2O2 induced an additional transient elevation of [Ca2+](i), which was abolished by the specific PLC blocker U73122 but was not eliminated by removal of extracellular Ca2+. PLC activity was increased by 1 mmol/L H2O2 but not by 400 micromol/L H2O2. CONCLUSIONS: H2O2 mobilizes Ca2+ through two distinct mechanisms. In one, 400 micromol/L H2O2-induced sustained [Ca2+](i) elevation is mediated via a Ca2+ influx mechanism, under which H2O2 impairs mitochondrial function via oxidative stress,reduces intracellular ATP production, and in turn opens ATP-sensitive, non-specific cation channels, leading to Ca2+ influx.In contrast, 1 mmol/L H2O2-induced transient elevation of [Ca2+](i) is mediated via activation of the PLC signaling pathway and subsequently, by mobilization of Ca2+ from intracellular Ca2+ stores.
Assuntos
Cálcio/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Peróxido de Hidrogênio/farmacologia , Oxidantes/farmacologia , Trifosfato de Adenosina/metabolismo , Amilorida/farmacologia , Animais , Inibidores Enzimáticos/farmacologia , Hepatócitos/citologia , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/fisiologia , Potenciais da Membrana/fisiologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Oxirredução , Técnicas de Patch-Clamp , Ratos , Ratos Wistar , Traumatismo por Reperfusão/metabolismo , Transdução de Sinais/fisiologia , Bloqueadores dos Canais de Sódio/farmacologia , Cianeto de Sódio/farmacologia , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/metabolismoRESUMO
Confocal microscopy and image analysis were used to compare driving forces, specificity, and regulation of transport of the fluorescent organic anion, Texas Red (sulforhodamine 101 free acid; TR), in lateral choroid plexus (CP) isolated from rat and an evolutionarily ancient vertebrate, dogfish shark (Squalus acanthias). CP from both species exhibited concentrative, specific, and metabolism-dependent TR transport from bath to subepithelial/vascular space; at steady state, TR accumulation in vascular/subepithelial space was substantially higher than in epithelial cells. In rat CP, steady-state TR accumulation in subepithelial/vascular spaces was reduced by Na(+)-replacement, but was not affected by a 10-fold increase in buffer K(+). In shark CP, Na(+)-replacement did not alter TR accumulation in either tissue compartment; subepithelial/vascular space levels of TR were reduced in high-K(+) medium. In both species, steady-state TR accumulation was not affected by p-aminohippurate or leukotriene C4, suggesting that neither organic anion transporters (SLC22A family) nor multidrug resistance-associated proteins (ABCC family) contributed. In rat CP, digoxin was without effect, indicating that organic anion transporting polypeptide isoform 2 was not involved. Several organic anions reduced cellular and subepithelial/vascular space TR accumulation in both tissues, including estrone sulfate, taurocholate, and the Mrp1 inhibitor MK571. In rat CP, TR accumulation in subepithelial/vascular spaces increased with PKA activation (forskolin), but was not affected by PKC activation (phorbol ester). In shark, neither PKA nor PKC activation specifically affected TR transport. Thus, rat and dogfish shark CP transport TR but do so using different basic mechanisms that respond to different regulatory signals.
Assuntos
Plexo Corióideo/metabolismo , Transportadores de Ânions Orgânicos/fisiologia , Squalus acanthias/metabolismo , Xantenos/farmacocinética , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Colforsina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Estrona/análogos & derivados , Estrona/farmacologia , Feminino , Técnicas In Vitro , Cinética , Leucotrieno C4/farmacologia , Masculino , Meglumina/farmacologia , Metotrexato/farmacologia , Modelos Biológicos , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Potássio/farmacologia , Propionatos/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Quinolinas/farmacologia , Ratos , Ratos Sprague-Dawley , Cianeto de Sódio/farmacologia , Ácido Taurocólico/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Xantenos/metabolismo , Ácido p-Aminoipúrico/farmacologiaRESUMO
Sodium cyanide-induced chemical hypoxia triggers a series of biochemical alterations leading to apoptosis in many cell types, including T cells. It is known that chemical hypoxia promotes inducible nitric-oxide synthase (iNOS) gene transcription by activating its transcription factors. To determine whether iNOS and NO production are responsible for chemical hypoxia-induced apoptosis, we exposed human Jurkat T cells to sodium cyanide in the presence or absence of iNOS inhibitors. We found that iNOS expression is necessary for hypoxia-induced lipid peroxidation and leukotriene B(4) generation. The inhibition of iNOS limited T-cell apoptosis by decreasing the activity of caspase-3 without affecting the expression of Fas/Apo-1/CD95 on the surface membrane of T cells. These data suggest iNOS-mediated NO produced endogenously in the T cell alters overall T-cell function and results in apoptosis. Proper control of iNOS expressed in the T cell may represent a useful approach to immunomodulation.